A Snapshot of Photoresponsive Liposomes in Cancer Chemotherapy and Immunotherapy: Opportunities and Challenges.

Introduction

The development and use of novel nanomaterials for theranostic (diagnosis and therapeutic) purposes is known as nanomedicine. 1 − 3 These nanoscale carriers promote the targeted controlled delivery of various therapeutic molecules via increasing endocytosis, improving tumor cellular uptake, and enhancing the permeability and retention effect (EPR). Furthermore, these nanocarriers improve the pharmacokinetic properties and half-lives of the different payloads while minimizing their toxic off-target impact on healthy tissues. 2 , 4 − 7

Liposomes are among the nanoscale carriers that represent well-known and reliable drug and gene delivery systems that are frequently employed in treatment of cancer, and the well-known liposomal doxorubicin (Doxil, Janssen Biotech, Inc.) represents the best example of a selective tumor targeting agent with decreased toxicity. It was the first FDA-approved liposomal medication for the treatment of several malignant tumors such as Kaponsi’s sarcoma, ovarian cancer, and multiple myeloma.

Liposomes that are triggered by external physical stimuli such as photoirradiation, magnetic fields, or X-ray irradiation represent novel nanocarrier systems that have an advantage over existing liposome drug delivery systems in that they allow on-demand payload release in response to external stimuli. 1 , 8 Using light as an external stimulus for different photoresponsive nanocarriers has several benefits compared to other external stimulants, such as noninvasiveness, sharp spatial resolution, simplicity, and affordability. 9 Light of an optimized wavelength is utilized as a stimulus for the photothermal therapy (PTT), photodynamic therapy (PDT), and controlled release of loaded drugs/nucleic acids from photosensitive nanosystems containing light-dissociable compounds, such as coumarin derivatives, o -nitrobenzyl, or others. 10 Upon exposure to light at the optimized wavelength, the nanostructures release their payloads via either the disassembly of the nanosystem by the photothermal effect, photocleavage, or the structural deformations of the nanosystem caused by photoisomerization. 9 Three light wavelength ranges are used as triggers: (i) ultraviolet (UV, 200–400 nm), visible light (400–650), and near-infrared (NIR, 650–900 nm). 11 Even though UV stimulation generates more effective photochemical reactions and potent energy that are effective in both photodynamic and photothermal therapies (PDT and PTT), NIR stimulation is commonly used for the reasons presented in Table 1 .

Lately, recent studies have exploited the advantages of both UV, such as low wavelengths, achieving high energy and effective photochemical reactions, and NIR, such as safety and deep tissue penetration irradiations. 12 In this regard, few strategies have been employed to convert NIR light to UV or visible light after passing the body tissues, such as using the two-photon absorption strategy and lanthanide-based nanostructures. 13 Visudyne (liposome for injection, Novartis AG) was the first to earn FDA approval in 2000 as a photoengineered liposome for the treatment of myopia and age-related macular degeneration. Verteporfin (VP) is a synthetic porphyrin that is activated to produce highly hazardous reactive oxygen species when exposed to light at 690 nm, causing local damage to the neovascular endothelium. 14

Immuno-oncology is the new era of cancer treatment. 15 − 17 It focuses mainly on exploiting this intrinsic tendency of the mammalian immune system to combat cancer. 18 − 20 The process of distinguishing cancer cells from healthy cells is known as immunosurveillance and is mainly carried out by the tumor-infiltrating lymphocytes such as adaptive cytotoxic T lymphocytes (CTLs) and innate natural killer cells (NK cells), 21 , 22 as shown in Figure 1 . However, despite the apparent immunogenicity of cancer, the body often fails to eradicate it or prevent metastasis on its own. This is partly due to the evident evolution of malignant cells, which evade the immune system by various mechanisms, including the exploitation of immune checkpoints and T cell exhaustion. 23

It has been evidenced that a liposome-based combination of phototherapy and immunotherapy can increase the therapeutic index of these modalities by improving the stability and biocompatibility of the cargo as well as minimizing the adverse effects. 24 , 25 In the same context, various studies have suggested that using nanocarriers in conjunction with immunotherapy and photothermal therapy could result in more significant antitumor immunological effects. 26 , 27

Although scientists have spent the last ten years focusing on light-triggered liposomes in drug/gene delivery, the shallow depth of light irradiation still limits the spectrum of therapeutic indications because the light is applied externally. Furthermore, if free medications are freely delivered, they are likely to be activated in nontarget sites. As a result, the authors in this Review address the most recent achievements in light-triggered liposomes with respect to their structure and encapsulation power for chemotherapeutic, immunotherapeutic, and oncological molecular targets, with a special emphasis on the potential alternative routes to these present obstacles for future research and clinical use.

Liposomes as a Drug Delivery System

Structure and Properties

Liposomes are developed by self-assembling biocompatible and biodegradable phospholipids and cholesterol in an aqueous medium to form concentric nanovesicles comprised of lipophilic tails (fatty acids chains) and polar heads (phosphate ester groups). 28

The unique structure of liposomes, an aqueous center entrapped within lipid bilayers, facilitates the chaperoning of various hydrophilic and hydrophobic therapeutic molecules such as phytochemicals, chemotherapeutic or immunotherapeutic agents, and RNA molecules for cancer therapy ( Figure 2 A). 4 , 29 , 30 Charged therapeutic moieties could bind to the liposomal membranes through electrostatic interactions ( Figure 2 A). Additionally, liposomes can cross the tumor cell membrane through endocytosis or membrane fusion ( Figure 2 B), expediting the preferential cellular uptake and passive accumulation of loaded drugs and/or photosensitizers (PS) inside cancer cells. 31 , 32 In addition, using liposomes as nanocarriers in cancer therapy is auspicious because their surfaces could be tailored with different targeting moieties, such as folic acid, hyaluronic acid, antibodies, and peptides, ensuring the active targeting of liposomes to their target site of action ( Figure 1 C). 33 , 34 Additionally, using liposomes as nanocarriers for drug/RNA/PS delivery would improve the pharmacokinetics of the loaded therapeutic molecules and prolong their half-lives (>72 h) via surface coating with polyethylene glycol (PEG, Figure 2 C).

The composition of the liposomes is crucial in controlling the integrity of the liposomes, as it gives the liposomes their unique features such as particle size, stiffness, fluidity, stability, and electrical charge. 35 , 36 The phospholipids (PLs) involved in designing liposomes might be natural, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), or synthetic, such as 1,2-dipalmitoyl- sn -glycero-3-phosphorylethanolamine (DPPE), 1,2-distearoyl- sn -glycero-3-phosphorylethanolamine (DSPE), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE), and 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC). In addition, cholesterol steroid (Chol) is used in combination with the PLs to control the fluidity of the liposomal membrane. Surfactants (Span 60 and Tween 60) are also used in the formulation of liposomes to lower the surface tension between various immiscible phases, improving the drug/PS encapsulation and release profiles. Finally, polymers, such as PEG (polyethylene glycol), are used to decorate the liposomal surface to shield the liposomes from circulating proteins, increasing systemic circulation time and lowering immunogenicity while increasing therapeutic efficacy. 35 The major components used in the formulation of liposomes and their chemical structures are summarized in Table 2 .

Phototriggered Liposomes (PTLs)

Design, Optimization, And Physicochemical Properties

Since the FDA approved the first photoresponsive liposomes, Visudyne, in 2000, several studies reported the engineering and optimization of light-sensitive liposomes loaded with various therapeutic moieties to achieve controlled and convenient cancer therapy. 37 , 38 Numerous physical and chemical properties should be considered when fabricating photoresponsive liposomes, as they can influence the drug encapsulation capacity, liposomal stability in body fluids, payload release percentage in response to light stimulation, and, eventually, the therapeutic activity. 39 These factors are lipid membrane fluidity, 40 lipid phase transition ( T m ), 41 and lipid polymerization. 42

Lipid membrane permeability is an essential factor that should be optimized to achieve the maximum payload release capacity upon exposure to a light stimulus. The components used in formulating PTLs are key players that control liposome’s physicochemical and biological features. For instance, using DSPC as one component of the PTLs led to an increase in the stability and entrapment efficiency of the liposomes as compared to using either dipalmitoylphosphatidylcholine (DPPC) or EPC. The decoration of the PTLs with the DPPE mPEG5000 polymer generates a steric hindrance around the liposomal membrane, minimizing the reticuloendothelial system uptake. Additionally, the inclusion of DPPG in the design of PTLs was reported to enhance the capability of the liposomes to penetrate the tumor cells due to its fusogenic features. 37 , 43 Finally, it was reported that increasing the concentration of Chol led to increasing the stiffness of the liposomal membranes while decreasing their fluidity. Additionally, increasing the molar ratio of Chol was found to reduce the membrane’s permeability. 41

The lipid phase transition ( T m ) is another critical factor affecting the cargo release from the lipid bilayer after light irradiation. It is affected by the composition of the phospholipids. Light illumination causes a photothermal effect, increasing the temperature above the T m .

Consequently, a phase transition of the lipid bilayer occurs from the orderly solid phase to the disassembled one, improving the selective diffusion of the loaded therapeutic moiety out of the liposomal membranes at the tumor site ( Figure 3 ). 41 For instance, a study reported the design of photoresponsive liposomes containing DPPC ( T m = 41 C) and gold nanoparticles. Upon irradiation with NIR radiation of 760 nm, the incorporated gold nanoparticles emit thermal energy (that exceeds the T m of DPPC) and thus lead to the release of the loaded drugs at the site of action. 44

Finally, lipid polymerization is another important property that affects the efficient release of loaded therapeutic agents upon light triggering. Photopolymerizable phospholipids, such as DPPC, are essential for disassembling the liposomal membranes, upon light irradiation, through the photo-cross-linking phenomenon. This leads to the selective release of the loaded drugs in the tumor tissues. 36

Payload Release Modalities from the Phototriggered Liposomes (PTLs)

The design of PTLs involves incorporating light-responsive entities (such as azobenzene) or PS (such as porphyrins derivatives) within optimized ratios of phospholipids using several approaches, such as thin-film hydration, ultrasonic dispersion, injection, freeze-drying, and microfluidic methods. 45 The typical structure of liposomes is exploited where the light-responsive molecules (PS) are loaded based on their water-solubility; the hydrophilic ones are encapsulated in the aqueous core, while the hydrophobic ones are incorporated in the lipid bilayer. 46 Upon light irradiation, the PTLs are disassembled via either physical or chemical activation of the incorporated light-responsive molecules, leading to the efflux of the payloads at the site of action. Table 3 summarizes the release modalities of the loaded therapeutic agents through either photophysical or photochemical activation.

The liposomes coloaded by PS and anticancer agents are accumulated in the tumor site via passive targeting through the enhanced permeability and retention (EPR) effect. In addition, PTLs could be actively targeted to the intended site of action by functionalizing their surfaces with target moieties that bind to specific overexpressed receptors in the tumor cells (folate receptors, for instance). Both passive and active targeting could decrease the off-target effects and help in the preferential accumulation of PS and payloads in the tumor tissues. Then, upon the light irradiation of the desired site of action, activating the PS leads to the PTT (or PDT) effects. 47

For instance, a study reported the design of GE11 peptide conjugated liposomes coloaded with curcumin, a natural chemotherapeutic, and indocyanine green (ICG), a photosensitizer. The presence of GE11 peptide facilitates the selective targeting of liposomes via binding to the epidermal growth factor receptor (EGFR) on the lung tumor surface. Then, upon NIR irradiation, the liposomes released their payloads, which synergistically generated reactive oxygen species (ROS) and induced apoptotic pathways, improving the anticancer effects while reducing off-target effects. 48

Phototriggered Liposomes in Cancer Chemotherapy and Immunotherapy

Phototriggered Liposomes Encapsulating Chemotherapeutic and/or Immunotherapeutic Agents

As previously mentioned, cancer nanomedicine is a hot topic that has been highly tackled by researchers from different disciplines. 55 This displays an arsenal of nanotherapeutics with proven better efficiency in the sustained delivery of several anticancer agents. 56 Phototriggered liposomes have been on top of the list of nanocarriers tailored for cancer patients, prompting the development of personalized nanomedicine among cancer patients. 57 , 58 PDT and PTT are less invasive local therapy methods that eliminate cancerous cells by producing highly toxic reactive oxygen species (ROS) or hyperthermia upon light illumination. 59 For instance, Hyp-HPbCD-loaded liposomes containing hypericin (Hyp) as a photosensitizer showed a phototoxic effect against ovarian adenocarcinoma only after irradiation. 60 Additionally, the encapsulation of indocyanine Green (ICG) in chitosan-coated liposomes gave stable liposomes, enhanced their uptake by B16-F10 melanoma cancer cells, and increased and improved phototoxicity. 61 According to another study, a novel multimodal delivery system using curcumin-loaded lipopolyplexes (LPPs) in combination with PDT was created, and it enhanced the delivery of RNA molecules to the SKOV-3 cancer cell. 62 In a different study, curcumin was loaded into liposomes made of HSPC and DPPE-mPEG5000, which improved the stiffness of the liposomal bilayer membrane, prevented curcumin from leaking outside the lipid membranes, and led to increased cytotoxicity toward cancer cells. 63 Another study reported the fabrication of photoresponsive liposomes that contained both the anticancer medication cisplatin and ICG. 64 Through a photothermal action mediated by ICG under NIR illumination, cisplatin was selectively released to tumor tissues, demonstrating an improved anticancer efficacy either with PTT alone or in conjunction with chemotherapy. 64 Furthermore, Wu et al.’s study described the creation of zwitterionic liposomes containing methylene blue (MB) PS, which may produce reactive oxygen species (ROS) in response to light exposure and induce the death of cancer cells. 65 This work encapsulated MB into zwitterionic liposomes to protect loaded medicines from degradation in the bloodstream, prolong systemic circulation, and improve MB cellular uptake into cancer cells. DSPC was used to self-assemble a zwitterionic polymer–lipid, poly(12-(methacryloyloxy)dodecyl phosphorylcholine), in a 1:4 molar ratio, which showed enhanced cytotoxicity on breast cancer cells through the release of ROS 65 ( Table 4 ).

The hypoxia caused by the tumor microenvironment is one of the issues that has been identified as an impeding factor for PDT’s effectiveness. Consequently, numerous methods have been devised to address these issues, such as coloading nanoparticles to provide adequate oxygen self-generation, which improved the effectiveness of PDT by reducing tumor microenvironment (TME)-induced hypoxia. Another idea is creating a unique liposomal platform encapsulating the catalase (CAT) enzyme for combinatory photo/chemotherapies to tackle tumor hypoxia. 66 For instance, doxorubicin (Dox), catalase, and NIR photosensitizer-loaded liposomes were invented with an improved lysosome targeting capability. Catalase reduced the amount of in the hypoxic TME and boosted the PDT’s efficacy by successfully causing the breakdown of H 2 O 2 to produce in situ O 2 . 66 Another intriguing work created novel light-triggered liposomes by coloading PS, chlorin e6, and the prodrug AQ4N, demonstrating a therapeutic potential in treating cancer under hypoxic conditions. 67 Under hypoxic conditions, the targeted delivered AQ4N was preferentially activated inside cancer tissues to release the more cytotoxic active form. This liposomal delivery system produced both the PDT effect and hypoxia-activated chemotherapy when exposed to LED light at 660 nm, which significantly improved the overall treatment results for the 4T1 tumor-bearing mouse model, as shown in Table 4 . 67 Moreover, to test the viability and potential of combining PTT and immunotherapy, PTT-responsive liposomes coloaded with the immunostimulatory drug polyinosinic:polycytidylic acid (poly I:C) and indocyanine green were formulated. 68 The liposomal structure was disassembled upon irradiation with 808 nm laser light, inducing a photothermal therapeutic impact against B16 melanoma tumor and CT-26 colorectal carcinoma models and accelerating cancer cell death. The released poly I:C from the liposomes clearly activates dendritic cells (DCs) in nearby lymph nodes.

Additionally, a new triple-sensitive liposomal system called NIL-IM-Lip (pH/MMP2/temperature triple-sensitive) was reported to remodel the repressed tumor lymph node immune microenvironment (TLIME) by simultaneously mobilizing the adaptive and the innate immune arms at the TME, namely, cytotoxic T cells and natural killer (NK) cells, respectively. 69 Following the pH-sensitive release of the NGR motif (that binds to CD13) and the MMP2-responsive release of IL-15, the temperature-sensitive NIL-IM-Lip is directed to the lymph nodes. In addition, it contains IR780 (photosensitizer) and 1-MT (IDO inhibitor) that simultaneously induce immunogenic cell death and suppress regulatory T cells during photothermal stimulation. 70

Phototriggered Liposomes Modulating Tumor Microenvironment and Cancer Cellular Behaviors through Encapsulating Genetic Material

The application of gene therapy to combat cancer oncological signaling pathways has been prompted by a growing body of knowledge in regard to its modulatory role for tumor cells and their surrounding TME. Gene therapy includes exogenous nucleic acids, such as genes, gene segments, oligonucleotides, microRNAs (miRNAs), or small interfering RNAs (siRNAs). Such genetic materials are delivered through ex vivo or in vivo modalities through several carriers, such as liposomes, nanoparticles, viral vectors, and many others. 72 In this section, the authors will shed light on the use of phototriggered liposomes in delivering genetic materials into cancer cells.

For instance, a study reported the usage of noncationic photoresponsive liposomes—Al(III) phthalocyanine chloride disulfonic acid (AlPcS2a)—and siRNAs ,where it was reported that the noncationic LPS results in minimizing off-target effects and improving the selective uptake of the siRNAs by cancer cells such as HepG2 and SK-HEP-1. 73 Several studies have recently reported the successful delivery of the CRISPER/Cas 9 system using liposomes. For example, the fabrication of liposomes loaded with photosensitizer verteporfin (VP) and sgRNA against the TNFAIP3 gene (that encodes the A20 protein, which is a tumor promoter in numerous cancer types, such as breast and liver cancers, and glioblastoma) was reported. The usage of these phototriggered liposomes resulted in a decrease in the TNFAIP3 mRNA level and increased knockdown efficiency. 74 Another multifunctional vehicle was reported for the delivery of Cas9-sgRNA. It was based on a lipid/AuNPs complex coated by TAT peptide for nucleus targeting and sgRNA targeting Plk-1 (a master mitotic regulator that is frequently overexpressed in tumor cells). This multifunctional system was successful enough to produce a PlK-1 knockout model. The designed liposomes led to effective tumor (melanoma) target gene (Plk-1) knockouts and tumor suppression owing to the synergic effect and the interaction between the photothermal-mediated intracellular release of the CP and the CP-induced cell death. 75 Table 5 summarizes the photoresponsive liposomes encapsulating genetic materials used in cancer therapy.

Phototriggered Liposomes As Carriers for Novel Immunotherapeutic Agents and TME-Remodeling Agents

Immune surveillance is the process by which different immune system elements safeguard the host against the establishment of primary tumors, promote tumor escape, or both, either by sculpting tumor immunogenicity or attenuating antitumor immune responses and forming what is called the TME. 15 , 78

Cancer immune editing or immune escape is the process by which cancer cells evade the immune system using many tactics, such as defective antigen presentation, inducing apoptosis for cytotoxic T cells and NK cells, immune suppressive mediators, immune checkpoints, and other mechanisms, and the best way to restore the antitumor effect of the immune system is by immunotherapy. 16 , 17

Various immunological elements are found upon cellular examination of a typical TME, indicating an immune battle against the tumor. 21 , 78 − 80 The appearance of multiple cytokines, macrophages, neutrophils, and tumor-infiltrating lymphocytes (TILs), among others, signify an attempt of the host immune system to eradicate the tumor. 22 , 81 − 84

As previously mentioned, the recent introduction of immunotherapeutic agents in the field of oncology has been considered a revolution that restored faith in many desperate cancer victims. 19 , 21 , 85 Cancer immunotherapy has significantly improved patients’ chances of survival and quality of life as compared to earlier standards of care (such as chemotherapy, radiation, and surgery), and there are many strategies for cancer immunotherapy like vaccination therapy, adoptive T cell therapy, and immune checkpoint inhibition on the molecular and cellular scale. 84 In this section, the authors will highlight the recent literature discussing the role of phototriggered liposomes in immunotherapy.

Liposome-Mediated Delivery of Immune Checkpoint Inhibitors

Immune checkpoints, by definition, are signals received by immune cells to either stimulate or repress their action. In a functional scenario, these direct the immune system to properly aggress during the initial stages of response before prompting it to regress during later stages to prevent autoimmune damage/prolonged inflammation. One example of an immune checkpoint dynamic is the interaction between the costimulatory T-cell receptor CD28 and the inhibitory cytotoxic T lymphocyte-associated antigen 4 (CTLA4), a receptor expressed in relatively low numbers only after T-cell activation. While CD28 prompts T-cell amplification upon binding to its B7 ligands (CD80 and CD86), CTLA4 counteracts this due to its higher affinity to the identical ligands and resultant competition with CD28, as shown in Figure 4 . 86

As the other key player in terms of immune checkpoints, PD-1 is a chief target of immune checkpoint blockade. A notable aspect of PD-1 is that its ligand, PD-L1, is commonly overexpressed on malignant cell surfaces to serve as another method for suppressive immune evasion method. The interaction between PD-1 and PD-L1 is characteristic of T-cell exhaustion and thus the abrogation of this interaction has been proposed to effectively “reverse” this exhaustion, as shown in Figure 4 . Henceforth, treatments by either anti-PD-1 mAb or anti-PD-L1 mAb have been tested and showed promising results. 87 , 88

Liposomes have been recently introduced to immune checkpoint blockade therapy, especially targeting PD-1/PD-L1 inhibitors, increasing their efficiency, and reducing their previously mentioned possible side effects. A double-layer system liposome has been employed by Lang et al. 89 to encapsulate the PD-1 inhibitor HY19991 and thioridazine. The liposomes successfully increased the accumulation of the HY19991 and thioridazine drugs inside the tumor tissue. 89

From another perspective, Merino et al. employed liposomes coupled with PD-L1 mAbs to modulate the immune system from the other side of the cancer–immune synapse. 90 Such approach results showed that liposomes containing 5% PEG showed the highest hindering of the PD-L1 suppressor effects. Furthermore, Hei et al. developed another type of liposomes coupled with anti-PD-L1 on their surface, yet they also contain the catalase enzyme inside them; those liposomes showed better therapeutic effects and, most importantly, low general toxicity. 91 Gu et al. also reported the reduced systemic toxicity and tissue damage of PD-L1 antibodies when formulated with liposomes in mice melanoma models. 92

It is worth noting that a combination of phototriggered liposomes and immune checkpoint inhibitors has been reported to be effective in improving the immunogenic profile of cancer cells. Lan et al. designed a new phototriggered liposome called Lp(DHA)@CP (composed of a polyunsaturated fatty acid-doped liposomal hydrogel) loaded with the PD-L1 antibody (αPD-L1) and photosensitizer chlorin e6 (Ce6). In response to the NIR light irradiation, abundant ROS were produced by the integrated Ce6, which then caused the liposomal hydrogel to disintegrate for the on-demand sustained release of PD-L1. Additionally, the produced ROS in TME was able to make low-immunogenic “cold” tumors “hot” by boosting the T cell infiltration in tumors, which in turn increased their potential responses to ICB treatments. 93

Another liposomal system coloaded with a photothermal agent (IR780), folic acid (FA) (that increases tumor targeting and also increases the enhanced permeability and retention (EPR) effect of the liposomal system), oxaliplatin (OXA), and PD-L1 inhibitors (BMS-1) was fabricated. The created liposomal system demonstrated advantageous tumor tissue biodistribution and tumor cell endocytosis, boosting the photothermal and anticancer effects, as well as improved dendritic cell maturation and infiltration of antigen-specific T lymphocytes into the residual tumors and blocking the immune checkpoint as summarized in Table 6 . 94

Liposome-Mediated delivery of Immunogene Therapy

Fusogenic liposomes represent an optimum RNA delivery vehicle, since they protect the RNA moiety from endocytic sequestration and lysosomal destruction. 95 Anionic fusogenic liposomes were created by Stremersch et al. and were assessed for their ability to transport siRNA to the monocyte/DC (JAWSII) cell line and B16F10 cancer cells. 96 Another interesting delivery method for DC targeting is RNA lipoplexes. When delivered systemically to lymphoid organs, they can shield RNA from extracellular ribonucleases and permit the selective expression of their RNA cargo in local APCs. By enhancing in situ CD8 T cell immunity, Salomon et al. revealed RNA lipoplexes encoding CD4 T cell-recognizable neoantigens and created powerful adaptive T cell responses. 97 Hybrid lipopolyplexes containing N 1-methyl pseudouridine nucleoside-modified mRNA has been reported by Thielemans et al. to diminish inflammatory responses without impairing T-cell immunity. Compared to mRNA and lipoplexes in this investigation, immunization with lipopolyplexes demonstrated robust T cell immunity and higher efficacy in suppressing tumor growth. 98

Phototriggered Liposomes: TME Remolding Agents

In addition to the conventional photosensitive liposomal effects in the ablation of tumors, PTT and PDT have been recently employed to alleviate the immunosuppressive TME intrinsically and, at the same time, act as a carrier for several immunotherapeutic agents. 99

On the mechanistic level, the produced heat during either PTT or PDT radiation can induce the eradication of cancer cells by activating the involved immune cells at the TME. This is mainly accompanied by the release of tumor-associated antigens, ATP, and high mobility group box 1 protein into the TME and the translocation of calreticulin to the cell surface. 100 Such release will result in the uptake of the antigens by antigen presentation cells and the activation of effector T cells and thus initiation signals for antitumor immunosurveillance episodes. 101 In addition, PTT-mediated intrinsic immunogenic effects can further enhance antitumor immune responses of immunotherapeutic agents, leading to combinational action for effectively eradicating cancer cells and alleviating the immune-suppressive TME. 102

Liu and colleagues formulated a photosensitive liposomal system coloaded with porphyrin as a photosensitizer and indoleamine-2,3-dioxygenase (IDO) (which is an enzyme that changes tryptophan (Trp) to kynurenine (Kyn), which could impair the survival and activity of CD8 + T cells) inhibitor as an immune-metabolic adjuvant. This designed liposomes were reported to initiate the cytotoxic T cell-mediated eradication of cancer cells through the induction of the immunogenic cell death (ICD) cascade and the simultaneous blockage of the IDO pathway. 103 This highlights the synergistic spectacles that could happen upon combining PDT and immunotherapeutic agents and the potent reversal of the immunosuppressive TME, fighting against metastatic cancers. 103 Shi et al. developed a liposome-based immune activation method that dramatically enhanced tumor growth inhibition when paired with photodynamic therapy. Specifically, they developed a novel liposomal-encapsulated catalase (CAT), lyso-targeted NIR photosensitizer (MBDP), and doxorubicin (Dox) to achieve combinatory chemo-PDT to promote tumor oxygenation and antagonize the immunosuppressive effects of the TME. 66

Another study by Wang et al. reported that a chlorine e6 (Ce6) liposome formulation encapsulating galectin-3 inhibitor low molecular citrus pectin (LCP) was used against melanoma by combining PDT with immunotherapy. 104 It was speculated that Ce6 induced PDT therapeutic effects against melanoma via apoptosis, inhibition of migration and invasion capacities of tumor cells, and eventually immune activation effects, as shown in Figure 5 . Kim et al. reported the potent immunomodulatory effects of light-triggered liposomes in a xenograft mouse model bearing human liver bile duct carcinoma cell line. 105 The Kim group formulated the liposomes using a PS-conjugated lipid encapsulating gemcitabine as a conventional chemotherapeutic agent. A confirmation of immunomodulatory cells at the TME recruited as a result of light-triggered liposomes has been done using immunohistochemical analysis and also helped in the maturation of dendritic cells, production of proinflammatory cytokines and, finally, activation of T cells. 105

Photoresponsive liposomes encapsulating immunotherapeutic and TME-modulating agents are summarized in Table 6 .

Challenges and Opportunities of Translational Phototherapy

Although various photoresponsive liposomes are considered the mainstay of phototherapy, poor light penetration limits the applicability to cutaneous conditions. 59 However, different solutions to this problem have been suggested, such as the creation of medical gadgets that make it easier to apply light endoscopically. In the case of Photofrin, an optic guide fiber diffuser was designed for employing endoscopically administered light to irradiate the lungs. 106

Another option is to construct photosensitizer in nanoformulation, limiting systemic circulation and managing the photosensitizer’s action. A phase 1/2 clinical trial ( NCT02367547 ) is currently being conducted to investigate using PDT against cancer utilizing a nanoscale lipid vesicle gel composition. 106

A phase 3 clinical trial is currently being conducted in primary liver cancer patients to assess the efficacy of combining microwave hyperthermia, radiofrequency, thermal ablation, or high-intensity focused ultrasound with ThermoDox/OPTIMA, a liposomal drug that releases DOX sealed in lysolipid thermally sensitive liposomes at temperatures above 40 °C. 37 , 107 , 108 This raises the question of whether the external stimulus that generates heat could be replaced with a light source, increasing the expandability of this nanoplatform.

Conclusions and Future Prospects

Despite the fact that phototriggered liposomes have been at the top of the list of nanocarriers that have prompted the development of precision cancer nanomedicine, there are still many challenges that limit the application of photoresponsive liposomes, particularly in solid tumors where the external shallow application of light may not be able to penetrate. This opens up the possibilities to new solutions, such as the development of nanoformulation photosensitizers that allow target-specific penetration or by embedding medical devices that allow for the use of endoscopically administered light.

This work is financially supported by the Special Research Account of the National and Kapodistrian University of Athens.

The authors declare no competing financial interest.

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